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The Streptococcus gordonii Adhesin CshA Protein Binds Host Fibronectin via a Catch-Clamp Mechanism*

  • Catherine R. Back
    Affiliations
    From the School of Oral and Dental Sciences, University of Bristol, Lower Maudlin Street, Bristol BS1 2LY, United Kingdom
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  • Maryta N. Sztukowska
    Affiliations
    the Department of Oral Immunology and Infectious Diseases, University of Louisville, Louisville, Kentucky 40202

    the Department of Dentistry, University of Information Technology and Management, 35-225 Rzeszow, Poland
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  • Marisa Till
    Affiliations
    the School of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom

    the BrisSynBio Synthetic Biology Research Centre, University of Bristol, Life Sciences Building, Tyndall Avenue, Bristol, BS8 1TQ, United Kingdom
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  • Richard J. Lamont
    Affiliations
    the Department of Oral Immunology and Infectious Diseases, University of Louisville, Louisville, Kentucky 40202
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  • Howard F. Jenkinson
    Affiliations
    From the School of Oral and Dental Sciences, University of Bristol, Lower Maudlin Street, Bristol BS1 2LY, United Kingdom
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  • Angela H. Nobbs
    Correspondence
    To whom correspondence may be addressed: School of Oral and Dental Sciences, University of Bristol, Lower Maudlin St., Bristol BS1 2LY, United Kingdom. Tel.: 441173424779
    Affiliations
    From the School of Oral and Dental Sciences, University of Bristol, Lower Maudlin Street, Bristol BS1 2LY, United Kingdom
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  • Paul R. Race
    Correspondence
    To whom correspondence may be addressed: School of Biochemistry, University of Bristol, Biomedical Sciences Bldg., University Walk, Bristol BS8 1TD, United Kingdom. Tel.: 44-1173311835
    Affiliations
    the School of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom

    the BrisSynBio Synthetic Biology Research Centre, University of Bristol, Life Sciences Building, Tyndall Avenue, Bristol, BS8 1TQ, United Kingdom
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  • Author Footnotes
    * This work was supported in part by National Institutes of Health Grants DE016690 (to H. F. J. and R. J. L.) and DE012505 (to R. J. L.), Biotechnology and Biological Sciences Research Council Grant BB/I006478/1 (to P. R. R.), and Royal Society University Research Fellowship Award UF080534 (to P. R. R.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
    ♦ This article was selected as one of our Editors′ Picks.
    3 The abbreviations used are: aaamino acidR13repeat region 13IDDintrinsically disordered domainSeMetselenomethionineSADsingle anomalous dispersionSAXSsmall angle X-ray scatteringc/pFncellular/plasma fibronectinNRnon-repetitiveIPTGisopropyl 1-thio-β-d-galactopyranoside.
Open AccessPublished:December 05, 2016DOI:https://doi.org/10.1074/jbc.M116.760975
      Adherence of bacteria to biotic or abiotic surfaces is a prerequisite for host colonization and represents an important step in microbial pathogenicity. This attachment is facilitated by bacterial adhesins at the cell surface. Because of their size and often elaborate multidomain architectures, these polypeptides represent challenging targets for detailed structural and functional characterization. The multifunctional fibrillar adhesin CshA, which mediates binding to both host molecules and other microorganisms, is an important determinant of colonization by Streptococcus gordonii, an oral commensal and opportunistic pathogen of animals and humans. CshA binds the high-molecular-weight glycoprotein fibronectin (Fn) via an N-terminal non-repetitive region, and this protein-protein interaction has been proposed to promote S. gordonii colonization at multiple sites within the host. However, the molecular details of how these two proteins interact have yet to be established. Here we present a structural description of the Fn binding N-terminal region of CshA, derived from a combination of X-ray crystallography, small angle X-ray scattering, and complementary biophysical methods. In vitro binding studies support a previously unreported two-state “catch-clamp” mechanism of Fn binding by CshA, in which the disordered N-terminal domain of CshA acts to “catch” Fn, via formation of a rapidly assembled but also readily dissociable pre-complex, enabling its neighboring ligand binding domain to tightly clamp the two polypeptides together. This study presents a new paradigm for target binding by a bacterial adhesin, the identification of which will inform future efforts toward the development of anti-adhesive agents that target S. gordonii and related streptococci.

      Introduction

      The adherence of bacteria to material and host cell surfaces is essential for colonization, persistence, and pathogenicity. This process is facilitated by bacterial cell-surface components termed adhesins, which recognize and bind specific partner molecules presented on the surfaces of host cells and other microorganisms (
      • Kline K.A.
      • Fälker S.
      • Dahlberg S.
      • Normark S.
      • Henriques-Normark B.
      Bacterial adhesins in host-microbe interactions.
      ). The filamentous adhesins, comprising pili and fibrils, are among the largest and most complex of all bacterial surface proteins. They are found in both Gram-positive and Gram-negative bacteria, including many pathogenic species, and have attracted considerable attention due to their roles in colonization, infection, and as vaccine candidates (
      • Demuyser L.
      • Jabra-Rizk M.A.
      • Van Dijck P.
      Microbial cell surface proteins and secreted metabolites involved in multispecies biofilms.
      ,
      • Löfling J.
      • Vimberg V.
      • Battig P.
      • Henriques-Normark B.
      Cellular interactions by LPxTG-anchored pneumococcal adhesins and their streptococcal homologues.
      ). Characteristically, these proteins form sizable polymeric assemblies that project outwards from the bacterial cell surface, presenting an adhesive target-binding region at their tip.
      Fibrillar adhesins are expressed by a wide variety of bacteria and display an extraordinarily diverse array of adhesive functions. In contrast to the extensively studied pili, much still remains to be learned about the structures and functions of these proteins. Fibrillar adhesins usually include a single multidomain polypeptide chain, covalently tethered to the bacterial cell wall (
      • Kline K.A.
      • Fälker S.
      • Dahlberg S.
      • Normark S.
      • Henriques-Normark B.
      Bacterial adhesins in host-microbe interactions.
      ). They frequently display exquisite selectivity for their partner ligands, with their modular architectures providing a platform for multifunctionality (
      • McNab R.
      • Forbes H.
      • Handley P.S.
      • Loach D.M.
      • Tannock G.W.
      • Jenkinson H.F.
      Cell wall-anchored CshA polypeptide (259 kilodaltons) in Streptococcus gordonii forms surface fibrils that confer hydrophobic and adhesive properties.
      ,
      • Larson M.R.
      • Rajashankar K.R.
      • Patel M.H.
      • Robinette R.A.
      • Crowley P.J.
      • Michalek S.
      • Brady L.J.
      • Deivanayagam C.
      Elongated fibrillar structure of a streptococcal adhesin assembled by the high-affinity association of α- and PPII-helices.
      • Gruszka D.T.
      • Wojdyla J.A.
      • Bingham R.J.
      • Turkenburg J.P.
      • Manfield I.W.
      • Steward A.
      • Leech A.P.
      • Geoghegan J.A.
      • Foster T.J.
      • Clarke J.
      • Potts J.R.
      Staphylococcal biofilm-forming protein has a contiguous rod-like structure.
      ). Although smaller than pili, they are inherently more complex, with divergent molecular structures, functions, and adhesive properties (
      • Jakubovics N.S.
      • Brittan J.L.
      • Dutton L.C.
      • Jenkinson H.F.
      Multiple adhesin proteins on the cell surface of Streptococcus gordonii are involved in adhesion to human fibronectin.
      ,
      • Jakubovics N.S.
      • Kerrigan S.W.
      • Nobbs A.H.
      • Strömberg N.
      • van Dolleweerd C.J.
      • Cox D.M.
      • Kelly C.G.
      • Jenkinson H.F.
      Functions of cell surface-anchored antigen I/II family and Hsa polypeptides in interactions of Streptococcus gordonii with host receptors.
      • Corrigan R.M.
      • Rigby D.
      • Handley P.
      • Foster T.J.
      The role of Staphylococcus aureus surface protein SasG in adherence and biofilm formation.
      ).
      Streptococcus species produce a multitude of fibrillar adhesins that promote binding to host cells and other microorganisms. These include, among others, the M proteins of Streptococcus pyogenes and the antigen I/II family polypeptides (
      • Fischetti V.A.
      Streptococcal M-protein–molecular design and biological behavior.
      ,
      • Jenkinson H.F.
      • Demuth D.R.
      Structure, function and immunogenicity of streptococcal antigen I/II polypeptides.
      • Rego S.
      • Heal T.J.
      • Pidwill G.R.
      • Till M.
      • Robson A.
      • Lamont R.J.
      • Sessions R.B.
      • Jenkinson H.F.
      • Race P.R.
      • Nobbs A.H.
      Structural and functional analysis of cell wall-anchored polypeptide adhesin BspA in Streptococcus agalactiae.
      ). Streptococcus gordonii, a primary colonizer of the human oral cavity and an opportunistic pathogen, has been shown to present a number of fibrillar adhesins on its surface, including the polypeptide CshA. In vitro and in vivo binding assays, gene disruption experiments, and heterologous expression studies in the non-adherent bacterium Enterococcus faecalis have established CshA as an important determinant of S. gordonii adherence (
      • McNab R.
      • Forbes H.
      • Handley P.S.
      • Loach D.M.
      • Tannock G.W.
      • Jenkinson H.F.
      Cell wall-anchored CshA polypeptide (259 kilodaltons) in Streptococcus gordonii forms surface fibrils that confer hydrophobic and adhesive properties.
      ,
      • McNab R.
      • Jenkinson H.F.
      • Loach D.M.
      • Tannock G.W.
      Cell-surface-associated polypeptides CshA and CshB of high-molecular-mass are colonization determinants in the oral bacterium Streptococcus gordonii.
      ). This protein has been shown to play a role in binding both to host molecules and a range of other microorganisms (
      • Holmes A.R.
      • McNab R.
      • Jenkinson H.F.
      Candida albicans binding to the oral bacterium Streptococcus gordonii involves multiple adhesin-receptor interactions.
      ).
      CshA is a 259-kDa polypeptide (
      • McNab R.
      • Forbes H.
      • Handley P.S.
      • Loach D.M.
      • Tannock G.W.
      • Jenkinson H.F.
      Cell wall-anchored CshA polypeptide (259 kilodaltons) in Streptococcus gordonii forms surface fibrils that confer hydrophobic and adhesive properties.
      ,
      • McNab R.
      • Jenkinson H.F.
      • Loach D.M.
      • Tannock G.W.
      Cell-surface-associated polypeptides CshA and CshB of high-molecular-mass are colonization determinants in the oral bacterium Streptococcus gordonii.
      ) that forms ∼60-nm peritrichous fibrils on the surface of S. gordonii (
      • McNab R.
      • Forbes H.
      • Handley P.S.
      • Loach D.M.
      • Tannock G.W.
      • Jenkinson H.F.
      Cell wall-anchored CshA polypeptide (259 kilodaltons) in Streptococcus gordonii forms surface fibrils that confer hydrophobic and adhesive properties.
      ). It shares <10% overall sequence identity to any protein of known structure, indicative of divergent or novel function. The CshA pre-protein is composed of 2508-amino acid (aa)
      The abbreviations used are: aa
      amino acid
      R13
      repeat region 13
      IDD
      intrinsically disordered domain
      SeMet
      selenomethionine
      SAD
      single anomalous dispersion
      SAXS
      small angle X-ray scattering
      c/pFn
      cellular/plasma fibronectin
      NR
      non-repetitive
      IPTG
      isopropyl 1-thio-β-d-galactopyranoside.
      residues, organized in the form of a leader peptide (residues 1–41), a non-repetitive region (residues 42–778), 17 repeat domains (R1–R17, each ∼101-aa residues), and a C-terminal cell wall anchor (
      • McNab R.
      • Jenkinson H.F.
      • Loach D.M.
      • Tannock G.W.
      Cell-surface-associated polypeptides CshA and CshB of high-molecular-mass are colonization determinants in the oral bacterium Streptococcus gordonii.
      ). CshA has been shown to bind the high molecular weight glycoprotein fibronectin (Fn) via an interaction that is mediated by the N-terminal non-repetitive region of the protein (
      • Jakubovics N.S.
      • Brittan J.L.
      • Dutton L.C.
      • Jenkinson H.F.
      Multiple adhesin proteins on the cell surface of Streptococcus gordonii are involved in adhesion to human fibronectin.
      ). Antibodies specific to this portion of CshA block Fn binding, whereas those raised against the repeat domain region elicit no effect (
      • McNab R.
      • Holmes A.R.
      • Clarke J.M.
      • Tannock G.W.
      • Jenkinson H.F.
      Cell surface polypeptide CshA mediates binding of Streptococcus gordonii to other oral bacteria and to immobilized fibronectin.
      ). The CshA-Fn interaction has been proposed to be of general significance in promoting S. gordonii colonization at a range of sites within the host (
      • Jakubovics N.S.
      • Brittan J.L.
      • Dutton L.C.
      • Jenkinson H.F.
      Multiple adhesin proteins on the cell surface of Streptococcus gordonii are involved in adhesion to human fibronectin.
      ,
      • McNab R.
      • Holmes A.R.
      • Clarke J.M.
      • Tannock G.W.
      • Jenkinson H.F.
      Cell surface polypeptide CshA mediates binding of Streptococcus gordonii to other oral bacteria and to immobilized fibronectin.
      ,
      • Giomarelli B.
      • Visai L.
      • Hijazi K.
      • Rindi S.
      • Ponzio M.
      • Iannelli F.
      • Speziale P.
      • Pozzi G.
      Binding of Streptococcus gordonii to extracellular matrix proteins.
      ). These include the cardiac endothelium, where adherence by S. gordonii is known to promote the onset of infective endocarditis, a severe, potentially fatal inflammation of the inner tissues of the heart (
      • Douglas C.W.
      • Heath J.
      • Hampton K.K.
      • Preston F.E.
      Identity of viridans streptococci isolated from cases of infective endocarditis.
      ).
      Despite the importance of CshA, little is known about the molecular structure and function of this protein. Such information would provide fundamental mechanistic insight and may inform the development of anti-adhesive agents that target S. gordonii and related streptococci.
      Here we report a structural and functional description of the non-repetitive Fn-binding region of CshA. We reveal that this part of the polypeptide is composed of three distinct domains, designated herein as non-repetitive domain 1 (NR1, CshA(42–222)), non-repetitive domain 2 (NR2, CshA(223–540)), and non-repetitive domain 3 (NR3, CshA(582–814)). In vitro Fn binding assays of truncated CshA proteins heterologously expressed on the surface of the non-adherent bacterium Lactococcus lactis demonstrate that both NR1 and NR2, but not NR3, confer adhesive properties to CshA. Biolayer interferometry analysis of Fn binding by the recombinant CshA NR domains reveals that NR2 binds both cellular and plasma fibronectin with significantly lower ka, kd, and KD values than NR1. Using circular dichroism (CD) spectroscopy, small angle X-ray scattering (SAXS), and allied biophysical methods, NR1 is shown to constitute a discrete intrinsically disordered domain (IDD). The crystal structure of NR2 is also presented, which adopts a lectin-like fold with a clearly identifiable ligand-binding site. Together, our data are consistent with a two-state mechanism of Fn binding by CshA, where NR1 functions to recognize and bind Fn, forming a dissociable pre-complex, which is subsequently stabilized by a high affinity binding interaction mediated by NR2. This “catch-clamp” mechanism of Fn binding may be of general significance in other bacterial adhesins that contain intrinsically disordered domains.

      Discussion

      In previous studies we have established the important role played by the S. gordonii cell wall-anchored polypeptide CshA in mediating adherence to host and microbial cell surfaces (
      • McNab R.
      • Forbes H.
      • Handley P.S.
      • Loach D.M.
      • Tannock G.W.
      • Jenkinson H.F.
      Cell wall-anchored CshA polypeptide (259 kilodaltons) in Streptococcus gordonii forms surface fibrils that confer hydrophobic and adhesive properties.
      ,
      • Jakubovics N.S.
      • Brittan J.L.
      • Dutton L.C.
      • Jenkinson H.F.
      Multiple adhesin proteins on the cell surface of Streptococcus gordonii are involved in adhesion to human fibronectin.
      ,
      • Holmes A.R.
      • McNab R.
      • Jenkinson H.F.
      Candida albicans binding to the oral bacterium Streptococcus gordonii involves multiple adhesin-receptor interactions.
      ,
      • McNab R.
      • Holmes A.R.
      • Clarke J.M.
      • Tannock G.W.
      • Jenkinson H.F.
      Cell surface polypeptide CshA mediates binding of Streptococcus gordonii to other oral bacteria and to immobilized fibronectin.
      ). Here we extend the scope of our investigations to include detailed molecular level characterization of the Fn-binding “non-repetitive region” of this protein. This portion of CshA possesses an aa sequence distinct from that of any polypeptide for which structural or functional studies have been conducted to date, indicative of novel form or function.
      In vitro cFn binding assays of heterologously expressed wild type CshA and truncated variants thereof confirm the critical role played by the non-repetitive region of CshA in facilitating cFn binding. These data also demonstrate that the adhesive properties of CshA are confined to the NR1 and NR2 domains of the protein. Surprisingly, a CshAΔNR1-expressing strain of L. lactis is more adherent to immobilized cFn than that expressing the wild type polypeptide. These data imply a mechanism of Fn binding by CshA where NR2 forms a dominant tight-binding interaction with its Fn substrate.
      Results from complementary experiments investigating the kinetics of Fn binding by recombinant NR proteins are consistent with our heterologous expression studies. NR1 is found to bind both cFn and pFn with a KD value in the low micromolar range. NR2 binds both forms of Fn with greater affinity than NR1, demonstrating that this domain forms a stable, less dissociable interaction with Fn than its NR1 neighbor. Conversely, NR1 exhibits both a ka and kd value for c/pFn greater than that of NR2, indicating that NR1 engages and disengages Fn more rapidly. NR3, which was found to be dispensable for Fn binding in our heterologous expression studies, is shown using the BLitz technique to bind cFn and pFn only weakly, indicating that this domain may not significantly contribute to Fn binding in vivo. Together, our data are consistent with a mechanism of Fn binding by CshA that involves a rapidly formed but readily dissociable NR1-Fn complex, in tandem with a higher affinity, but less frequently formed NR2-dependent interaction.
      To further probe the mechanism of Fn binding by CshA, recombinant NR1 and NR2 were subjected to structural characterization. NR1 is shown to exhibit all the hallmarks of an IDD, including aberrant hydrodynamic behavior and an absence of secondary structure (
      • Habchi J.
      • Tompa P.
      • Longhi S.
      • Uversky V.N.
      Introducing protein intrinsic disorder.
      ,
      • Uversky V.N.
      A decade and a half of protein intrinsic disorder: Biology still waits for physics.
      ). SAXS analysis of NR1 demonstrates that the protein adopts a disordered but partially compacted structure in solution. Such a conformation is typical of IDDs, which form loosely assembled dynamic structures, stabilized by residual local and long-range weak intramolecular forces (
      • Uversky V.N.
      Functional roles of transiently and intrinsically disordered regions within proteins.
      ). This results in inherent conformational adaptability and, in the case of IDDs that are involved in protein or ligand binding, confers an expanded capture radius, enabling target binding over greater distances than can be achieved by globular proteins (
      • Huang Y.
      • Liu Z.
      Kinetic advantage of intrinsically disordered proteins in coupled folding-binding process: a critical assessment of the “fly-casting” mechanism.
      ). Target engagement by IDDs may be concomitantly accompanied by partial or complete recovery of a fold state, a process that often brings both partners into closer proximity, potentially enhancing the rate of binding by the so-called “fly-casting” mechanism (
      • Shoemaker B.A.
      • Portman J.J.
      • Wolynes P.G.
      Speeding molecular recognition by using the folding funnel: the fly-casting mechanism.
      ). Given that the energy needed to recover secondary structure is abstracted from the interaction energy of binding, IDDs generally form low affinity interactions with their binding partners, characterized by remarkably fast on- and off-rates (
      • Kiefhaber T.
      • Bachmann A.
      • Jensen K.S.
      Dynamics and mechanisms of coupled protein folding and binding reactions.
      ). Fn binding by NR1 exhibits many of these defining characteristics, including rapid complex formation and dissociation and a weak yet specific relative binding affinity. Interestingly, NR1 is devoid of sequence motifs that have been previously implicated in IDD-mediated bacterial adherence to cell-surface molecules, implying that Fn binding by CshA NR1 may proceed via a mechanism that is distinct from, for example, the tandem β-zipper model (
      • Schwarz-Linek U.
      • Werner J.M.
      • Pickford A.R.
      • Gurusiddappa S.
      • Kim J.H.
      • Pilka E.S.
      • Briggs J.A.
      • Gough T.S.
      • Höök M.
      • Campbell I.D.
      • Potts J.R.
      Pathogenic bacteria attach to human fibronectin through a tandem β-zipper.
      ). There was no evidence of NR1-NR2 complex formation, as assessed by native PAGE, when the two domains were mixed in vitro, implying that NR2 does not bind NR1 and act to template its folding.
      In contrast to NR1, the NR2 domain of CshA is shown to adopt a globular structure with a lectin-like fold. The protein presents a clearly identifiable ligand-binding site on its surface that is populated by a combination of aromatic and negatively charged residues. This composition is consistent with a role for NR2 in the binding of carbohydrates or glycoproteins, a function further supported by our in vitro binding data, and the identification of structural homologues of NR2 that mediate protein-carbohydrate interactions (
      • Nylander Å.
      • Svensäter G.
      • Senadheera D.B.
      • Cvitkovitch D.G.
      • Davies J.R.
      • Persson K.
      Structural and functional analysis of the N-terminal domain of the Streptococcus gordonii adhesin Sgo0707.
      • Forsgren N.
      • Lamont R.J.
      • Persson K.
      Crystal structure of the variable domain of the Streptococcus gordonii surface protein SspB.
      ,
      • Troffer-Charlier N.
      • Ogier J.
      • Moras D.
      • Cavarelli J.
      Crystal structure of the V-region of Streptococcus mutans antigen I/II at 2.4 angstrom resolution suggests a sugar preformed binding site.
      • Cid M.
      • Pedersen H.L.
      • Kaneko S.
      • Coutinho P.M.
      • Henrissat B.
      • Willats W.G.
      • Boraston A.B.
      Recognition of the helical structure of β-1,4-galactan by a new family of carbohydrate-binding modules.
      ,
      • Back C.R.
      • Douglas S.K.
      • Emerson J.E.
      • Nobbs A.H.
      • Jenkinson H.F.
      Streptococcus gordonii DL1 adhesin SspB V-region mediates coaggregation via receptor polysaccharide of Actinomyces oris T14V.
      ). The ligand-binding site of NR2 is capped by a sizeable loop, for which no electron density was observed during structure elucidation. The absence of compelling electron density in this region is indicative of flexibility. It therefore appears likely that the capping loop plays a role in gating access to the NR2-binding site, potentially acting to expose this highly charged pocket during Fn engagement. Based on available structural and functional data, it is not possible to unambiguously identify the specific glycosylation(s) upon Fn that are bound by NR2, although this represents a major focus for future investigations.
      In summary, we report herein a structural and functional dissection of the Fn-binding, non-repetitive region of the S. gordonii fibrillar adhesin CshA. Uniquely, CshA appears to employ a bipartite mechanism of target binding that involves the coordinated action of a low affinity but high capture radius IDD, in partnership with a high affinity, high specificity ligand binding domain. Our data suggest a two-step catch-clamp Fn-binding mechanism, wherein the disordered NR1 domain of CshA acts to engage and “catch” Fn, via a process that is expedited by its elongated, disordered conformation. The resulting NR1-Fn pre-complex is rapidly formed and dissociated, although it may be stabilized via an NR2-mediated tight binding interaction, which functions to “clamp” CshA and Fn together (Fig. 7). Such a mechanism offers an elegant solution to the problem of forming highly specific intermolecular interactions within molecularly rich environments such as the human host. A number of fibrillar adhesins have been identified that incorporate IDDs, and as such the catch-clamp mechanism outlined herein may be of general applicability to other bacterial surface proteins that possess such domains (
      • Moschioni M.
      • Pansegrau W.
      • Barocchi M.A.
      Adhesion determinants of the Streptococcus species.
      ). Finally, the unusual target binding mechanism exhibited by CshA, along with molecular insights provided herein, presents a framework for the development of new anti-adhesive interventions that target disease causing streptococci and related bacteria. Such interventions could be based on multicomponent agents that target both the IDD and ligand-binding steps during adherence.
      Figure thumbnail gr7
      FIGURE 7Schematic depicting the catch-clamp mechanism of Fn binding by CshA. The intrinsically disordered NR1 domain of the protein rapidly engages and binds Fn in a process expedited by the sizable capture radius of the domain. Fn binding results in the formation of a dissociable pre-complex that may be accompanied by the recovery of NR1 secondary structure. The resulting pre-complex is stabilized by a high affinity binding interaction mediated by the NR2 domain of CshA.

      Author Contributions

      R. J. L., H. F. J., A. H. N., and P. R. R. conceived and designed the study. C. R. B., M. N. S., M. T., A. H. N., and P. R. R. performed experiments and analyzed data. C. R. B., M. N. S., R. J. L., H. F. J., A. H. N., and P. R. R. wrote the manuscript. R. J. L., H. F. J., and P. R. R. acquired funding. All authors reviewed the results and approved the final version of the manuscript.

      Acknowledgments

      We thank Jane Brittan and Lindsay Dutton for technical assistance, Dr. Christopher Arthur for mass spectrometry analysis, and Kristian Le Vay, Genevieve Baker, and Dr Steven Burston for fruitful discussions.

      Author Profile

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